To date, studies from our laboratory have shown that the strongest predictor of excessive alcohol intake is the response of the HPA Axis, with high levels of cortisol and ACTH being highly predictive of alcohol intake. Chronic activation of the neuroendocrine stress axis via high alcohol intake is also thought to contribute to allostatic changes at the level of the brain, which ultimately can lead to alcoholism. Corticotropin-releasing hormone (CRH) is the primary neuropeptide responsible for activation of the HPA axis and a potential candidate gene for alcohol abuse problems. Dr. Schwandt investigated age-dependent variation in the effects of acute ethanol administration on HPA-axis function and central monoamine turnover in adolescent rhesus macaques. After controlling for rearing condition, males (but not females) showed a positive correlation between age and alcohol-induced ACTH levels, and a negative correlation between age and alcohol-induced cortisol levels. For the monoamine metabolites, males and females showed similar negative correlations between age and percent change from baseline in alcohol-induced HVA and age and percent change from baseline in alcohol-induced MHPG. Females (but not males) also showed a significant negative association between age and percent change from baseline in alcohol-induced 5-HIAA. These results suggest that both age and sex are important factors when investigating the effects of ethanol on HPA axis activity and central nervous system functioning. Dr. Christina Barr completed sequencing the CRH gene and promoter region in rhesus. Among the variants that she reported last year is a SNP (-248C>T) that is present within a highly-conserved region critical to regulation of CRH gene transcription. This year, she performed in vitro functional assays, demonstrating that, while there is no effect of this allele under basal conditions, there is an increase in reporter activity in TPA-treated cells expressing the -248T construct. This is consistent with findings in last year?s report, in which she reported the ?248 T polymorphism to be associated with increased ACTH and cortisol responses to social separation stress in infant macaques. She also found there to be an interactive effect with early rearing, in that ACTH and cortisol levels are especially high among carriers of the -248T allele that exposed to early adversity in the form of parental deprivation. Dr. Barr assessed another rhCRH promoter gene variant (-2232 C>G) which she showed disrupted one of the glucocorticoid response elements (GREs). This response element is known to be critical to feedback control of CRH expression by corticosteroids. This variant is in allelic identity with 18 other polymorphisms spanning the rhCRH gene, producing a specific haplotype cluster (rhCRH-A2). New in vitro studies showed decreased sensitivity to glucocorticoids in cells expressing the rhCRH-A2 construct. Unlike the CRH?248T polymorphism, the rhCRH-A2 haplotype is associated with increased basal, but not stress-induced, levels of ACTH. This rhCRH-A2 haplotype variant is associated with nonstress, mother-infant interactions. Infants who were solicitous of maternal contact (i.e., high infant approach and social contact with their mother, as well as low maternal rejection and infant behavioral withdrawal) were more likely to possess the rhCRH-A2 haplotype variant. There was also an association between the rhCRH-A2 haplotype variant and the infant?s independence of its mother (infrequent maternal restraint but high infant rejection of mother, vocalizations and play with peers). This was particularly true among female infants. The rhCRH-A2 haplotype is also associated with increased HPA response to alcohol, and adolescent animals carrying the rhCRH-A2 haplotype exhibit increased alcohol consumption with consecutive exposures, consistent with the putative role of CRH and the HPA axis in promoting escalation of alcohol and drug intake. As with the effects of this haplotype on infant behaviors, its effects on alcohol response and alcohol consumption are more pronounced among females. When Dr. Barr analyzed the effect of this variant on alcohol response, she found that female carriers of the rhCRH-A2 haplotype variant also exhibit lower levels of sedation and ataxia following alcohol infusion. In our continued collaboration with Dario Maestripieri and Kai McCormack maternal maltreatment was assessed in abusive and normal control mother-infant pairs. Subjects were genotyped for the serotonin transporter genotype. Infants with the l/s genotype demonstrated deviations in social play and higher rates of anxiety. In response to stress, l/s infants were more behaviorally reactive, and mounted a larger cortisol response than l/l infants. Similarly, l/s mothers mounted a larger cortisol response to separation from their infants. There were also complex rearing by genotype interactions. When compared to the other groups, abused infants with the l/s genotype demonstrated higher levels of resting cortisol during month 1, when abuse rates were highest, but over the next few months demonstrated consistently lower levels of cortisol than all other subjects. These results suggest that the short 5-HTT allele is associated with increased behavioral and physiological reactivity to stress, and that genotype may also modulate the effects of maltreatment during development. Dr. Schwandt and her team examined the HPA response of adult females who had been differentially reared as infants. As infants the subjects were reared as controls with their parents (MR), without adults but constant social experience with same aged peers (PR), or with limited social experience as surrogate-peer-reared (SPR) monkeys. SPR monkeys are provided with a terrycloth-covered surrogate from birth but unlike peer-reared (PR) subjects are limited in their social experiences to a period of one to three hours per day. 3-5 hears following these early rearing experiences, these adults, now mothers, were tested for plasma cortisol levels at baseline, during a social separation stressor, and during a recovery phase. SPR mothers had significantly lower levels of plasma cortisol than adult MR or PR mothers but only during recovery following the social separations. There were no differences between groups at baseline or during separations. These results suggest the early experiences affect an adult mother?s capacity to recover from stress. Work from Kagan and colleagues at Harvard show that children who are withdrawn and shy are at risk for anxiety disorders and alcohol abuse. A large group of rhesus monkeys (N = 71) were characterized using a social separation stressor as high, moderate, and low in behavioral withdrawal, defined using baseline, homecage withdrawal behaviors. Highly withdrawn animals showed less reduction than easygoing animals in CSF serotonin metabolite concentrations over repeated separations. Unlike their easygoing counterparts, highly withdrawn macaques failed to adapt and attenuate their stress-related plasma cortisol concentrations across separation weeks. Studies are planned this year to characterize these subjects alcohol intake. Depression and impaired CNS serotonin functioning are risk factors for excessive alcohol intake. In a collaboration with Carol Shively at Wake Forest, we found associations between hippocampal 5HT1a receptor binding and CSF 5-HIAA concentrations in nondepressed monkeys. These relationships were not apparent in depressed monkeys. Depressed monkeys showed higher 5HT1a receptor binding in the dentate gyrus. These observations suggest that behavioral depression in monkeys may have neurobiological substrates similar to human depression, and that impaired CNS serotonin may explain the commonality between alcoholism and depression.